1. Endogenous Nuclear RNAi Mediates Behavioral Adaptation to Odor
Bi-Tzen Juang, Chen Gu, Linda Starnes, Francesca Palladino, Andrei Goga, Scott Kennedy, Noelle D. L’Etoile 
Cell 
Volume 154, Issue 5, Pages (August 2013)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
HPL-2 and MUT-7 Act at the Time of Odor Exposure in the AWC Neurons to Promote Adaptation to Butanone
(A) Olfactory adaptation paradigm. Animals exposed to buffer alone (naive) or butanone (adapted) for 80 min are placed at the “origin” of an agar-lined 10 cm Petri dish. Butanone is placed at the red and ethanol at the black “X.” Sodium azide (to paralyze the worms) was also placed at each “X.” Animals roam plates 2 hr before counting. The CI is calculated by subtracting the number of animals at the diluent from the number at the odor and dividing this by the number of animals that left the origin.
(B) Initial screen of mutant strains defective for siRNA pathways. Bars represent mean CIs of strains of the indicated genotype that had either been incubated with buffer (−) or buffer-diluted butanone (+) for 80 min. Bars for wild-type represent the mean CI of pooled controls for all the strains. All error bars are SEM. The side-by-side comparisons of each strain with wild-type controls are shown in Figure S1A. ∗∗ = p < 0.005, ∗ = p < 0.05, and “n.s.” = p > Unless otherwise noted, all tests were two-tailed Student’s t test, and all assays were performed on separate days with >100 animals per assay. drsh-1, alg-2, dcr-1: n = 4; rde-1 mut-7: n = 6; hpl-2: n = 5.
(C) HPL-2 and MUT-7 are expressed in AWCs. Fluorescent confocal images of wild-type animals expressing the putative hpl-2 (top) or mut-7 (bottom) promoters driving GFP-tagged versions of each protein. AWC is marked with ceh-36prom3 promoter driving mCherry. Anterior is at the left for both images. Figure S1B is associated with this panel.
(D) Expression of HPL-2 or MUT-7 in AWC rescued the adaptation defects of each mutant. HPL-2 (left graph, third pair of bars) or GFP-tagged MUT-7 (right graph, third pair of bars) was expressed in AWC from pceh-36prom3 in hpl-2(tm1489) or mut-7(pk204), respectively. ∗∗p = 0.002, ∗p = , and n > 5 for each.
(E) Expression of HPL-2 or MUT-7 at the time of odor exposure rescued adaptation defects. hpl-2(tm1489) (left) or mut-7(pk204) (right) transgenic for the respective cDNA under the control of the heat shock promoter (phsp16-2) were heated (+) 1 hr before odor exposure. Heat-treated animals’ exposed CI’s were significantly different from either before heating (p = 0.02, hpl-2; p < 0.005, mut-7) or from nontransgenic animals that had been heated (p = 0.005, hpl-2; p < 0.005, mut-7), n > 5 for each.
(F) HPL-2 and MUT-7 act in the same genetic pathway for adaptation. Mean naive (−) and exposed (+) CIs of animals of the indicted genotype. The adaptation defects of the fbf-1(ok91) strain are due to loss of the translational control pathway (Kaye et al., 2009) that acts in parallel with hpl-2.
(G) Expression of NRDE-3 in AWC rescued the adaptation defects of the nrde-3 mutant strain. Mean CI of naive (−) and exposed (+) wild-type, ndre-3(gg66), and NRDE-3 expressed in AWC (pceh-36prom3) of the nrde-3(gg66) mutant strain. Figure S1D is associated with this figure.
Error bars for each panel are SEM.
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4. Figure 2
Prolonged Odor Stimulation Dynamically Regulates odr-1-Derived 22G RNAs, Association of HPL-2 with the odr-1 Locus, and Levels of odr-1 mRNA
(A) Diagram of the odr-1 and unc-40 genes. The odr-1 and unc-40 22GRNAs examined are indicated with arrows below the gene. The PCR amplicons for ChIP-qPCR are in green. PCR amplicons for mRNA analysis are in red.
(B) Prolonged odor exposure decreases odr-1 mRNA levels. Bars represent the mean fold change in unc-40 (gray) or odr-1 (black) mRNA level as a function of odor exposure (adapted/naive). RNA from animals of the indicated genotype was normalized to act-3 mRNA. The red line indicates “no change,” and the significance of the difference between a sample and “no change” was assessed using a two-tailed Wilcox signed rank test. ∗∗ indicates that median of sample and “no change” are different; p < # indicates a difference of p = (nonparametric, pair-wise comparison) in medians between the naive and adapted values of the mRNA. p values displayed are from two-tailed Mann-Whitney test of medians. Chemotaxis behavior for each population and the individual data points for each pair are shown in Figure S2A. Error bars represent SEM, and n > 3.
(C) Chemotaxis behavior correlates with the level of odr-1 mRNA in butanone-adapted animals. The butanone CI of odor-exposed animals was compared with their odr-1 mRNA level (mRNA levels normalized to act-3 mRNA). Red circles indicate wild-types, and blue triangles indicate mut-7(pk204) animals expressing MUT-7 solely within the AWC neurons. r is Pearson’s correlation coefficient, and p is from a two-tailed Student’s t test (wild-type, n = 8; AWC MUT-7 rescue, n = 5).
(D) Prolonged odor exposure increases NRDE-3-bound odr-1 22GRNA levels. The first five bars represent mean fold change in total 22GRNAs normalized to odor-insensitive sn2343 RNA in adapted versus naive animals of the indicated genotype. Error bars represent SEM. Red line indicates no change. ∗ = p < 0.03, Wilcoxon signed-rank test for median values versus no change. p values displayed are the comparison of medians using an unpaired two sample Mann Whitney nonparametric t test, n > 3. Figure S2B is associated with this panel. The last three bars represent mean fold change in pnrde-3::NRDE-3 coIPed 22GRNA (n = 6) normalized to the odor-insensitive X-cluster. Error bars represent SEM. ∗ = p < 0.04, Wilcoxon signed rank test for median values versus no change. Displayed p values are from a pairwise, one-tailed t test, p = of medians. Figure S2C shows the behavior.
(E) Prolonged odor exposure specifically increases HPL-2 binding to the odr-1 locus in a MUT-7-dependent fashion. The mean ratio of 3XFLAG-HPL-2 expressed in AWC (podr-3) coimmunoprecipitated odr-1 (dark bars) or unc-40 (light bars) in adapted versus naive animals is shown above the genotype of each population. Error bars represent SEM. Also indicated is the PCR-amplified, coIPed region of each locus corresponding to “A,” “B,” and “C” in (A). Coimmunoprecipitated DNA from each locus was normalized to input. This was then normalized to the ratio of IPed act-3 to input. act-3 levels were odor insensitive. ∗p = 0.031, one-tailed Wilcoxon signed-rank test comparing median values to no change (the red line). The median value of odr-1 B was compared to unc-40 B; p = using a two-tailed Mann Whitney test. n = 5. The final set of bars represents background from nontransgenic animals. Figure S2D shows the behavior.
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5. Figure 3 HPL-2 and MUT-7 Act Downstream of Nuclear EGL-4
(A) Current model for long-term olfactory adaptation of the AWC neuron. Acute stimulation of AWC localized G-protein-coupled receptors (GPCR) by odor (left) causes animals to chemotax toward the odor. After prolonged odor exposure (right), the cGMP-dependent protein kinase (PKG) EGL-4 translocates to the nucleus to cause animals to ignore the odor for prolonged periods of time.
(B) Once in the nucleus, EGL-4 requires HPL-2 and MUT-7 to promote adaptation. The chemotaxis index of the indicated strains that express constitutively nuclear EGL-4 from a transgene (+) were compared to their siblings that did not carry this transgene (−). rde-2 is a control, adaptation-proficient strain (Figure S1A). Importantly, all animals were naive to butanone. n > 3 with >100 animals analyzed per assay. ∗∗p < , two-tailed Student’s t test. Bars represent the mean CIs, and the error bars represent SEM.
(C) HPL-2 and MUT-7 are not required for odor-induced nuclear entry of EGL-4. GFP-tagged EGL-4 was expressed in either wild-type, hpl-2(tm1489), or mut-7(pk204) strains. Animals were exposed to buffer alone (naive) or butanone for 80 min before imaging. The percentage of the population that showed nuclear EGL-4 in one AWC neuron was determined.
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6. Figure 4
Phosphorylation of HPL-2 and MUT-7 at Predicted PKG Sites Is Required for Adaptation
(A) Schematic of HPL-2 and MUT-7. (Top) HPL-2 contains an N-terminal chromodomain, a C-terminal chromo shadow domain, and four predicted PKG phosphorylation sites. (Bottom) MUT-7 contains two predicted functional domains—cytosolic fatty acid binding domain and 3′ to 5′ exonuclease—and seven predicted PKG phosphorylation sites.
(B) Phosphorylation of predicted PKG target sites in MUT-7 is required for adaptation. Mean CIs of wild-type or mut-7(pk204) strains expressing the indicated form of MUT-7 in AWC. Figure S3D shows individual lines. n = 3 and p value is from a two-tailed Student’s t test. The lines rescued the sterility defects of mut-7(pk204) (Figure S3B).
(C) Phosphorylation of HPL-2 at predicted PKG sites is required for adaptation. CIs of animals of the indicated genotype that expressed the indicated form of HPL-2 cDNA in AWC are shown. n > 3 with >100 individuals per assay. p value is from an unpaired Student’s t test.
(D) Nuclear EGL-4 phosphorylates HPL-2 in vitro. (Left) 3XFLAG-nuclear EGL-4 kinase was immunopurified from worms (behavior in Figure S3F) and incubated with purified HPL-2 and 32P ATP. The reactions were resolved on a gel and stained with Coomassie blue as loading control (lower) followed by autoradiography (upper). (Right) Quantification of five independent kinase assays. 32P phosphorylated HPL-2 was normalized to Coomassie stained band. Values shown for mutant HPL-2 substrate are shown as fold reduction of phosphorylation relative to HPL2-wild-type, which was set to 1. Error bars represent mean ± SEM (p < ; two-tailed Student’s t test, n = 5).
(E) Phosphorylation of HPL-2 at a predicted PKG phosphorylation site in the CSD is sufficient to decrease butanone chemotaxis in naive animals. CIs of wild-type animals expressing the indicated form of HPL-2 in AWC, n > 3. Figure S3E shows CIs of individual lines. All strains expressed similar levels of the indicated transgenes as assessed by GFP intensity. p values are from a two-tailed Student’s t test.
(F) Phosphorylation of HPL-2 at the EGL-4 phosphorylated sites is sufficient to promote adaptation in naive animals. CIs of naive wild-type, osm-9, or mut-7 animals either expressing the phosphomemetic HPL-2(S/Tto E) (+) or not (−). In all panels of this figure, the bars represent the mean values, and the error bars represent SEM; n > 5. p value is from an unpaired two-tailed Student’s t test.
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7. Figure 5
Prolonged Stimulation Induces Long-Term Olfactory Adaptation in the AWC Neurons via an siRNA and Chromatin-Remodeling-Dependent Process
Model for how prolonged butanone stimulation may lead to long-lasting olfactory adaptation in the AWC neuron. Asterisks indicate processes and factors shown to act in AWC. Odor exposure stimulates a seven transmembrane GPCR at the cell membrane and causes EGL-4 to enter the nucleus where it phosphorylates HPL-2 (solid arrow) and may also phosphorylate MUT-7 (dashed arrow). Phosphorylated HPL-2 promotes adaptation in a 22GRNA dependent process by binding to H3K9me3 (yellow flags). Phosphorylated MUT-7 boosts levels of odr-1 22G RNAs. These siRNAs act as guides within the NRDE-3 Ago complex to direct H3K9me3 to odr-1 gene. Phosphorylated HPL-2 would repress transcription of the odr-1 gene (red inhibitory bar). Finally, lower levels of odr-1 mRNA decreases the animal’s attraction to butanone.
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8. Figure S1
Examining RNAi Defective Strains for Butanone Adaptation, Related to Figure 1
(A) A genetic screen for RNAi defective strains that are also butanone adaptation defective. This is related to Figure 1B and Table 1. Bars represent mean butanone chemotaxis indices of animals that were pre-exposed to either S-basal buffer alone (light gray bars) or butanone diluted in S-basal buffer (dark gray bars) for 80 min before being subjected to butanone chemotaxis assays. The indicated strain was compared to a wild-type control that was grown and assayed in parallel on the same day; error is the SEM. Differences between the CIs of adapted wild-type and mutants were analyzed by paired two-tailed Student t test with equal variance (∗ indicated < 0.05 and ∗∗ indicated < 0.005). dcr-1(ok247) and drsh-1(ok369) are marked with hT2::GFP(I, III). The animals were assayed on independent days with > 50 animals per assay. dcr-1(ok247), n = 4; rde-4(ne337), n = 3; eri-1(mg366), n = 6; drh-1(tm1329), n = 4; drh-2(ok951), n = 4; drh-3(ne4253), n = 6; rrf-1(pk1417), n = 7; rrf-2(ok210), n = 4; rrf-3(pk1426), n = 8; rde-1(ne300), n = 4; ergo-1(gg98), n = 3; quintuple MAGO, n = 3; MAGO12, n = 3; rde-2(pk1657), n = 4; rde-3(3364), n = 3; mut-7(pk204), n = 4; nrde-3(gg66), n = 4; nrde-2(gg91), n = 5; nrde-1(gg88), n = 5; hpl-2(tm1489), n = 5; hpl-1(tm1624), n = 3; drsh-1(ok369), n = 4; alg-2(304), n = 5.
(B) Odor does not affect the subcellular localization of HPL-2 or MUT-7, related to Figure 1C. (Top) GFP tagged HPL-2 cDNA (short form) was expressed under 3.9 kb of the promoter region in the hpl-2(tm1489) mutant background. The transgenic animals were incubated with S Basal buffer alone (naive; left) or S Basal buffer containing butanone (adapted; right) for 80 min before imaging. The AWC neuron was marked by expression of podr-1::DsRed and HPL-2 was only observed in the nucleus. No change in the compact nuclear appearance of GFP tagged HPL-2 was observed after odor-exposure. (Bottom) GFP fused MUT-7 cDNA was expressed under the AWC specific promoter (pceh-36prom3) in mut-7(pk204) mutant strains. The confocal images of naive (upper) and odor-exposed (lower) transgenic animals were captured. MUT-7 was expressed throughout the entire AWC neuron and this was not altered with odor exposure.
(C) Individual lines expressing NRDE-3 in AWC rescue the adaptation defects of nrde-3(gg66). This figure is related to Figure 1G. The mean CIs of naive and butanone pre-exposed strains are shown. nrde-3(gg66) mutant strains carrying transgenic arrays expressing NRDE-3 under the AWC specific podr-3PROM3 were tested alongside wild-type and nrde-3(gg66) parental strains. All transgenic lines were created by independent injections. Differences between adapted animals of nrde-3(gg66) and the individual transgenic lines were analyzed by two-tailed t test. ∗ indicates p < 0.05 and ∗∗ indicates p < Line 4 is shown in Figure 1G.
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9. Figure S2
Changes in odr-1-Derived RNA Species in Adaptation, Related to Figure 2
(A) Prolonged odor exposure decreases odr-1 mRNA levels, related to Figure 2B. (Top) Bars represent the mean chemotaxis indices for the populations that were used in the odr-1 mRNA analyses in Figures 2B and 2C. Each chemotaxis assay was performed with > 100 animals that had been removed from the rest of the population before it was processed for RNA. (Bottom) Pairwise comparison between the normalized expression level of odr-1 mRNA (odr-1 mRNA molecules per act-3 mRNA molecule) from buffer exposed (naive, squares) and odor exposed (adapted, triangles) populations. A single population of animals that were grown at the same time was split in half and used in the paired analysis. The genotypes compared are wild-type; mut-7(pk204) and mut-7(pk204);Expceh-36prom3::MUT-7. Of note, act-3 mRNA levels were not altered by odor exposure. To allow comparison between wild-type and mut-7 as well as the AWC-specific rescue of mut-7, the y axis for the middle and right pairs was expanded; the units remain the same (odr-1 mRNA/act-3 mRNA). P values were obtained by using a one-tailed Wilcox signed rank test of paired medians.
(B) Prolonged odor exposure increases odr-1 22G RNA, related to Figure 2D. (Top) Bars represent the mean chemotaxis indices for the populations used in the odr-1 22G RNA analyses in Figure 2D (bars 1-5). As can be seen, pAWC::GFP::MUT-7 rescued the mut-7(pk204) strain’s adaptation defects. (Bottom) Pairwise comparison of levels of odr-1 (odr-1.7 and odr-1.6) unc-40 (unc-40.2) derived 22G RNAs from population of animals that showed adaptation to butanone. Expression of 22G RNA was quantified by quantitative real-time PCR with a Taqman probe and normalized to an endogenous sn2343 gene whose levels did not change with odor treatment. The normalized expression of each butanone-exposed population (triangles) was compared with its buffer-exposed (squares) control that was grown at the same time and on the same plates and was part of the same initial population. A line links the 22G values of each paired population. Different genotypes are compared: left, wild-type pairs; middle, mut-7(pk204) pairs, right, mut-7(pk204);Expceh-36prom3::MUT-7 (MUT-7 expressed in AWC) pairs. To allow comparison between wild-type and mut-7 as well as the AWC-specific rescue of mut-7, the “y” axis for the middle and right pairs was expanded; the units remain the same (odr-122G RNA/107 sn2343). Note: the two very high values for odr-1siRNA in graph #4 from mut-7(pk204) resulted when we used miRNeasy Micro kit (QIAGEN) rather than the isopropanol precipitation that was used for the other samples.
(C) Olfactory behavioral assay results for populations that were analyzed for NRDE-3 co-IP odr-1 22G RNA, related to Figure 2D (bars 6-8). Bars represent the mean chemotaxis indices for the populations that were used in the NRDE-3 associated odr-1 22G RNA analyses in Figure 2D (bars 6-8). This shows that expression of 3XFLAG tagged NRDE-3 in wild-type animals does not alter their olfactory behavior.
(D) Bars represent the mean chemotaxis indices for the populations that were used in Figure 2E in which podr-3::3X FLAG::HPL-2 was expressed in wild-type or mut-7(pk204) animals. podr-3 drives expression in AWB, AWC and AWA neurons and this expression does not alter behavior in wild-type or mut-7(pk204) animals. HPL-2 associated DNA was immunoprecipitated from the populations indicated. ∗ indicates two-tailed t test p < 0.05 for adapted CIs in wild-type and mut-7(pk204) animals with transgenes.
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10. Figure S3
Phosphorylation of HPL-2 and MUT-7 at PKG Consensus Sites Is Required for Adaptation, Related to Figure 4
(A) Alanine substitutions of all PKG consensus sites fail to rescue mut-7(pk204) adaptation defects. Bars represent the mean CIs of butanone naive and exposed animals of the indicated genotypes. mut-7(pk204) mutant animals expressing wild-type MUT-7 under the control of 3.2 kb of the mut-7 promoter region (pmut-7) restored adaptation, while mut-7 mutants failed to adapt to butanone when they expressed pmut-7::MUT-7(all 7 PKG sites mutated into alanine) (compare 3rd and 4th pairs of bars). Error bars represent the SEM. p values are from two-tailed Student’s t test.
(B) Alanine substitutions of all PKG consensus sites rescue the brood size defects of mut-7(pk204). Bars represent the mean he number of eggs produced by 9 hermaphrodites of the same transgenic lines in Figure S3A were counted. Animals were kept at 25°C and brood size was determined in 3 independent experiments. The defects of the brood size in mut-7 mutant backgrounds were partially rescued to the same extent whether wild-type MUT-7 or MUT-7 with alanine mutations was expressed (compared 3rd to 4th bars). Bars represent means and error bars are SEM, p values are from two-tailed Student’s t test.
(C) Individual lines expressing MUT-7(S/T all A) mutants in mut-7(pk204) all fail to rescue the adaptation defects of mut-7. All lines were tested in parallel with wild type and mut-7(pk204). The mean value of CIs from the number of independent assay days marked above each strain are represented by the bars. Error bars represent the SEM. p values are from two-tailed Student’s t test.
(D) A genetic screen for PKG sites within MUT-7 that are required for adaptation - related to figure 4B. Bars represent the mean butanone chemotaxis index of wild type (N2) animals expressing various forms of GFP-tagged MUT-7 from the AWC specific pceh-36PROM3 that had been exposed to buffer (light bars) or butanone in buffer (dark bars) for 80 minutes before the assay. The PKG consensus sites in MUT-7 that were changed from serine (S) or threonine (T) to alanine (A) are indicated below each group of bars. Each group of bars represents independent lines. (-) indicates N2 without a transgene. When all sites were changed (all S/T to A) 3/5 lines failed to adapt as determined by the adapted CI being significantly higher than N2. The lines that had a naive CI lower than 0.5 are not included in the analysis. Thus, 1/2 lines expressing MUT-7(T11A), 0/6 lines expressing MUT-7(T236A), 0/7 expressing MUT-7(T383A), 0/6 expressing MUT-7(S516A), 0/4 expressing MUT-7(T549A) and 2/2 expressing MUT-7(T656A) and 6/9 MUT-7(S883A) failed to adapt to butanone. This suggests that MUT-7 is likely to require phosphorylation at these sites to function in adaptation. Each line was analyzed n≥2 with >100 adult animals each assay. Error bars represent the SEM. p values are from two-tailed Student’s t test. ∗ indicates p<0.05 of two-tailed Student t-test between butanone exposed wild type and transgenic animals and ∗∗ indicates p<0.005 significant difference.
(E) A genetic screen for PKG sites within HPL-2 that are sufficient to induce adaptation-like behavior in the absence of odor- related to 4E. All or four individual serines (S) and threonines (T) within the HPL-2’s PKG sites were changed to glutamic acid (E) in the GFP-tagged HPL-2a cDNA. 20 ng/μl of each plasmid was introduced into wild-type animals and individual transgenic animals deriving from independent injections were analyzed for their ability to chemotaxis towards butanone. The bars represent the mean CIs of >3 independent days for each indicated line. Several lines carrying HPL-2(T29E) or HPL-2(T104E) show mild chemotaxis defects, while two lines with HPL-2(S155E) show more severe chemotaxis defects. The PKG site at S155 in the chromo shadow domain is likely to play a key residue for EGL-4 phosphorylation. Two-Tailed t-test were performed by analyzing CIs of unexposed wild type and transgenic animals from at least three independent assays each line and ∗ indicates p<0.05 and ∗∗ indicates p<0.05. The lines used in Figure 5E were: all, line 1; T29E, line 2; S82E, line 4; T104E line 3; S155E line 4. Error bars represent the SEM. p values are from two-tailed Student’s t test.
(F) 3XFLAG::NLS::EGL-4 is active as judged by its ability to promote adaptation in naive animals, related to Figure 4D. 2XNLS FLAG tagged EGL-4 were expressed in wild type animals. These naive animals were analyzed for their chemotaxis to butanone. Bars represent the mean values from >3 independent assays. As can be seen, animals expressing nuclear EGL-4 caused chemotaxis defects. The gain-of-function animals were harvested and EGL-4 was purified from whole worm extracts by FLAG immunoprecipitation. These extracts were used in vitro kinase assays in Figure 4D.
(G) HPL-2 is phosphorylated at consensus PKG sites by a commercial bovine cGMP-dependent protein kinase. We wanted to determine whether the PKG consensus sites in HPL-2 that we had identified as being important for adaptation are substrates for a well characterized, purified kinase rather than being the result of phosphorylation by a kinase that might co-purify with 3XFLAG-EGL-4. To do this, we incubated 2 ng of PKG1α (EMD Millipore) with 1.5 micrograms of wild type or mutant HPL-2 in our standard assay. Graph is the quantification of two independent kinase assays. The error bars represent mean ± SEM (p = 0.03; two-tailed t test). The amount of phosphorylated 32P ATP substrates were normalized to their respective Coomassie stained bands. Values shown for mutant HPL-2 substrate are shown as fold reduction of phosphorylation relative to HPL2-WT set to 1. At least half the signal in wild type represents phosphorylation at the identified PKG sites, indicating that these sites are bona fide PKG phosphorylation sites.
(H) EGL-4 is the only PKG required for adaptation of the AWC chemosensory response, related to figure 4. C09G4.2 was predicted to translate a cGMP-dependent protein kinase (PKG) in C. elegans. This second C. elegans PKG is called pkg-2. To investigate if PKG-2 acts in the AWC neurons, two deletion alleles, ok966 and tm3878, were analyzed for their ability to adapt to butanone. pkg-2(ok966) and pkg-2(tm3878) each were able to adapt to butanone as well as the wild type strain. All error bars represent SEM and assays were performed on ≥ 3 separate days with > 100 animals per assay. A GFP reporter construct with 2 kb upstream of the predicted ATG start codon of pkg-2 is expressed in >15 head neurons, none of which was AWC neurons (A. Gupta and N.D.L., unpublished data).
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